1. The Field of the Invention
This invention relates to devices used to analyze blood. More particularly, the present invention relates to devices-used to assess platelet function in blood.
2. The Background Art
Platelets are among the smallest corpuscular components of human blood, having a diameter 2-4 .mu.m. The identification of platelets as a class of blood corpuscles was described as early as 1882 with the importance of platelets for the formation of a hemostatic plug or clot being first reported about 1888. Another milestone in knowledge about platelets was reached around 1925 when two important concepts in hemostasis (curtailing bleeding or hemorrhaging) were expressed: Aggregations of platelets as they are present in a platelet plug which stops bleeding can only be formed as long as the blood is flowing; and, Formation of fibrin is not a primary event in thrombosis, but is preceded by important changes of the corpuscular elements of the blood. The importance of platelets in the clotting of blood is now well-known.
The number of platelets in a healthy human typically varies from 150,000/mm.sup.3 to 300,000/mm.sup.3 of blood. While platelets are commonly referred to as cells (and will also be referred to in this disclosure as "cells"), strictly speaking platelets are not cells since they do not have a nucleus. Platelets are produced by the bone marrow, where megakaryocytes (as the results of mitotic proliferation of a committed progenitor cell) liberate platelets as the end product of protrusions of their membrane and cytoplasm. The typical shape of resting platelets is discoid and upon activation they undergo a shape change to a globular form with pseudopodia (up to 5 .mu.m long) which facilitates the formation of clots.
In modern medical practice, a variety of parameters inform clinicians of the condition of a patient. No surgeon, internist or anaesthetist would treat a patient without assessing liver function, renal function, blood coagulation, blood count and electrolytes before an elective surgical intervention or as follow up in critically ill patients. However, even though a clinician desires an accurate report on blood platelet function, the existing tests do not adequately quantify blood platelet function.
The devices used to quantify blood platelet function are generally referred to as platelet aggregometers. Such platelet aggregometers attempt to evaluate the function of blood platelets and have been in use for many years. Clinical laboratories test for blood platelet function when there is reasonable suspicion that platelet function may be impaired in a patient. Similar tests are also performed on research samples to test the efficacy of various platelet-modifying agents.
Prior methods of assessing platelet aggregation contain inherent flaws related to (1) the testing of the platelets in an unnatural environment (platelets are assessed in an altered sample), (2) the requirement of significant technician time (and associated costs), and (3) the risk of exposing the technician to contact with the blood sample (and the risk of transferring blood-borne pathogens). As explained below, the most common method of assessing platelet aggregation is the optical transmittance method, which requires separating all other blood cells from the platelets. It is conventional wisdom in the industry that separation of platelets from other blood components does not significantly change the behavior of platelets but, as recognized by the present invention, the erythrocytes likely affect the platelet aggregation process and their removal results in an analysis of platelet function in an unnatural environment. Additionally, the procedure for separating the other cells from the platelets is also expected to separate a sub-population of platelets from the sample to be tested, and the removal of that sub-population may further distort the analysis of platelet aggregation. Moreover, the removal of non-platelet cells from the sample requires operator time and exposure of the operator to the risk of contact with any blood-borne pathogens.
One previously available type of existing platelet aggregometer utilizes optical transmittance. Also referred as the turbidometric method, the optical transmittance aggregometers are based on the technique of detection of light transmitted through a cuvette containing platelet-rich plasma (PRP). This technique was originally introduced by Born (Born, G. V. R., Nature, 194: 927-929, 1962) and optical transmittance aggregometers, and variations thereof, are described in U.S. Pat. Nos. 3,989,382 and 4,135,818, 4,066,260 (use of a rotating-disk cuvette), U.S. Pat. No. 5,325,295 (performing the measurement in microwells), U.S. Pat. No. 5,563,041 (adding an inhibitor to more completely prevent fibrin formation), and U.S. Pat. No. 5,569,590 (pre-mixing the needed reagents in a visual-detection system).
As platelets form aggregates, the light transmission through the blood increases in proportion to the aggregation response. The optical transmittance method attempts to detect the shape change, the rate of aggregation, the size of the aggregates, and the maximum aggregation of platelets. However, the recorded responses (except when dose-response techniques are used) can only be qualitative and offer little information relating to the number and size of the platelet aggregates formed. A major limitation of the optical transmission/turbidometric method is that it only works with PRP. Therefore, not only does the optical transmissive/turbidometric method entail time-consuming processing of blood (centrifugation to obtain PRP), but cells which may be potentially relevant to platelet aggregation (erythrocytes, leukocytes and certain subpopulations of platelets) are removed from the test sample during the centrifugation of the blood sample. All optical transmittance aggregometers disadvantageously require that the blood sample undergo centrifugation to separate leukocytes and red blood cells from the platelets. Centrifugation always results in the loss of some platelets and the previously available devices merely ignore the loss and its effect on quantitative measurements of platelet function. During centrifugation, larger platelets may sediment with the red cells and the effect of their removal on platelet-aggregation tests may well be significant.
Moreover, optical transmittance aggregometers disadvantageously require sample preparation and handling by a trained technician. Moreover, optical transmittance aggregometers disadvantageously assess the platelet function under unnatural conditions, that is in an altered blood sample.
Furthermore, as suggested earlier, preparation of platelet-rich plasma for testing using an optical transmittance aggregometer, as well carrying out the platelet aggregation test, requires manual pipeting and handling of blood by a technician. The manual handling of blood samples is not only time consuming but also exposes the technician to the risk of contact with a blood-borne pathogen. Additionally, red blood cells are known to affect the dynamics of platelet aggregation and with optical transmittance aggregometers using PRP the aggregation is assessed in the absence of red blood cells, thus eliminating possible important variables in the test for abnormal platelet function.
Another device and method for assessing platelet function utilizes the changing electrical impedance of the blood sample. With the electrical impedance method, electrodes are placed in the blood sample to monitor changes in the impedance after a platelet agonist is added to the sample. The electrical impedance method is described in Riess, H., Am. J. Clin. Pathol., 85: 50-56, 1986 and U.S. Pat. No. 4,319,194. The electrode surfaces and electrical effects used in the electrical impedance aggregometer may disadvantageously contribute to the platelet response and aggregation so that the response of the platelets to the aggregating agent alone which is added to the blood sample is not clear when electrical impedance aggregometers are used. In fact, measurements made with electrical impedance aggregometers show no reversal of platelet aggregation under conditions where such reversal is seen in other aggregometers (those using light-transmission measurements in PRP). Disadvantageously, this has led researchers to believe that platelet aggregation in whole blood is irreversible under essentially all conditions, which may not be true.
The electrical impedance method does not require the removal of blood cells from the sample, but dilution of the whole blood sample, generally by 50%, increases measurement sensitivity, which also alters the environment from the natural one in which the platelets normally reside. In addition, the electrical impedance method requires that the platelets adhere to the electrode surfaces immersed in the blood sample.
The impedance method detects the changes in electrical impedance caused by the deposition of activated platelets or platelet aggregates onto two electrodes submerged in the blood sample. The changes in impedance have been shown to correlate positively with platelet aggregation as detected by turbidometric methods. Unlike the turbidometric method, the impedance method allows for the measurement in merely diluted blood. Thus, the impedance method requires less manipulation of the blood sample than the optical transmission method. However, there are several discrepancies between the observations with the impedance method and those with the optical transmission/turbidometric method. When using the impedance method, shape change, disaggregation and biphasic aggregation response are not detectable and platelet response to epinephrine is extremely poor. Moreover, it has been reported that the inhibition of aggregation by antagonists such as aspirin and prostagndin was not normally detected when using the impedance method even though the inhibition of platelet release occurred.
Thus, the impedance method relies upon the adhesion of platelets to the metallic electrodes and is therefore only an indirect measure of platelet aggregation which occurs in the fluid phase. Importantly, the behavior of platelets adhering to an electrode is likely very different than the behavior of platelets adhering to one another in unaltered blood. Furthermore, as with other previously available methods and devices, the blood sample must be transferred to the electrode chamber and then removed again, and the electrodes must be cleaned and reused, both of which require technician time and also exposing the operator to the risk of contact with the blood and any pathogens contained therein.
Yet another method and device which is available for platelet testing is the immobilized platelet stimulant aggregometer. The immobilized platelet stimulant aggregometer measures the adhesion of platelets to an immobilized platelet stimulant. The blood sample or PRP sample is placed in a chamber in which some walls are rotating and some are stationary in order to produce a shear field. The stationary walls are coated with an agent which stimulates platelet adhesion and aggregation, and the rate of platelet response is monitored by a number of means, such as observing the adhesion through the transparent wall or measuring light transmission through the blood (when using PRP). The immobilized platelet stimulant method is described in U.S. Pat. No. 5,523,238 but has not gained widespread acceptance in clinical use.
Yet other lesser used techniques which have been used to measure platelet aggregation include the luminescence method and the platelet counting method. The luminescence method monitors the release of ATP from the dense granule by a firefly luciferin-luciferase assay in whole blood or PRP. In the platelet-counting method, the number of platelets in the test medium is counted intermittently during aggregation. Neither the luminescence method nor the platelet-counting method are considered common laboratory techniques for assessment of platelet aggregation.
In view of the forgoing, it would be an advance in the art to provide a method and apparatus for measuring platelet aggregation which accurately reflects platelet behavior in platelet's normal native environment, including whole blood samples. It would also be an advance in the art to provide an improved method and apparatus to assess platelet aggregation function which utilizes light scattering techniques rather than light transmissive techniques. It would be a further advance in the art to provide a method and apparatus for measuring platelet aggregation which minimizes technician contact with a blood sample being tested and which provides a test which can be carried out quickly and accurately.